Figure 4.

Time-of-drug-addition assay to identify the target of kaempferol. (A) Schematic diagram of the assay. (B) HFFs were pre-treated with kaempferol, acyclovir, or DMSO for 3 h and inoculated with VZV-YC01 at a MOI of 0.1. After virus inoculation for 1 h, drugs were removed, and HFFs were incubated for 72 h (pre-treatment). In addition, HFFs were inoculated with cell-associated VZV-YC01 at a MOI of 0.1 and treated with kaempferol (15 µg/mL), acyclovir (5 µM), or DMSO at 0, 6, 12, 18, 24, and 36 h after inoculation. Cells were harvested 72 h after inoculation, at which point total DNA was extracted and the relative amount of viral DNA was analyzed by qPCR using primers specific for open reading frame 62 (ORF62) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Experiments were performed in triplicate and GAPDH-normalized values were expressed relative to those in cells treated with DMSO (defined as 100%). * p < 0.005, ** p < 0.05 (Student’s t-test).